We sought to identify and characterize naturally occurring deletion mutants in internal genes of influenza virus so that they could be evaluated for their ability to confer attenuation. For example, persistent expression of influenza virus polymerases in permissive cells transfected with cloned influenza DNA should provide a system for complementation and selection of viruses containing mutations in the polymerase genes. The recently developed shuttle vector system that contains a mutant DHFR gene as a selectable marker was employed. Recombinant DNA containing separate promoters for transcription of the influenza PB2 DNA and DHFR DNA was constructed and used for transfection of simian CV-1 cells. The transfected cells were exposed to methotrexate for selection of transfected cells. Methotrexate-resistant cells will be evaluated for stable expression of the PB2 polypeptide by biochemical analysis.